The precise balance between excitatory and inhibitory neurotransmission is critical for healthy brain function. In the mature mammalian brain, inhibitory signaling is predominantly accomplished through the neurotransmitter -aminobutyric acid (GABA) binding to ligand-gated chloride (Cl-) channels (GABAA receptors). In contrast, in the immature brain GABAergic neurotransmission is excitatory in order to promote trophic activities necessary for development. The strength and polarity of GABA-mediated neurotransmission is determined by the intracellular chloride concentration. Changes in the relative activities of two chloride cotransporters, NKCC1 and KCC2, are responsible for the developmental switch in GABAergic signaling from excitatory to inhibitory. Importantly, disruptions in cellular chloride homeostasis resulting from dysfunctional NKCC1 and/or KCC2 activity result in neuronal hypo- or hyperexcitability that is implicated in the pathogenesis of epilepsy, chronic ischemia, and neuropathic pain. An understanding of the mechanisms responsible for regulating NKCC1 and KCC2 activity is lacking and is critical to the development of novel therapeutic strategies that target diseases resulting from altered Cl- homeostasis. While long-term differences in cotransporter activity are likely mediated by changes in gene expression, there is increasing evidence for acute regulation through post-translational modification and membrane trafficking. Protein palmitoylation is a reversible post-translational modification known to regulate various aspects of neuronal protein trafficking and function. The proposed research expands upon the preliminary finding that both NKCC1 and KCC2 are palmitoylated to test the hypothesis that palmitoylation regulates cotransporter activity either by modulating their protein expression, maturation, oligomerization, phosphorylation, trafficking, and/or chloride transport. Inhibitor and mutational approaches to prevent cotransporter palmitoylation will be applied in various biochemical and cell biological assays to test the effect of palmitoylation on cotransporter biology. To determine if developmental changes in cotransporter activity correlate with changes in palmitoylation, the acyl- biotin exchange (ABE) method, which was developed by our laboratory to quantitatively assess protein palmitoylation, will be used to characterize the palmitoylation of endogenous NKCC1 and KCC2 at various stages of brain development. Lastly, the ABE method in conjunction with knockdown and dominant negative approaches will be used to identify the enzymes responsible for mediating cotransporter palmitoylation. In summary, this work will explore a potentially novel mechanism for the regulation of NKCC1 and KCC2 activity and could provide insight into various pathologies that result from altered Cl- homeostasis as well as identify novel therapeutic strategies.